The basic objectives of this research are (1) to determine details of the internal structure of nucleosomes, the structural subunits of chromatin, such as the order of histone binding along DNA and the locations of specific histone-histone and histone-DNA contact sites; and (2) to investigate how non-histone proteins, histone modifications, and histone subspecies variations alter the basic structure of nucleosomes or chromatin in relation to active gene structure. Presently we are exploring the uses of staphylococcal protease, which recognizes acidic amino acids, for probing the regions of histone chains that are on the accessible surface of the nucleosome and for identifying sites of specific crosslinks or chemical modifications of histones. In addition we are studying in detail the small fraction of chromatin that is solubilized when mouse myeloma or chicken erythrocyte nuclei are digested slightly with micrococcal nuclease and equilibrated with near physiological saline solution. Such chromatin fractions contain nucleosomes that are highly enriched in the High Mobility Group and many other non-histone proteins. Present evidence suggests that these fractions are enriched in transcribed gene sequences and DNa originating from the vicinity of replication forks. Electrophoretic methods have been developed for separating these nucleosomes into species that differ in non-histone protein content. Work in progress is designed to determine the relationships between specific non-histone proteins and specifci gene sequences or DNA functions.